Quantitation of Oxidative Modifications of Commercial Human Albumin for Clinical Use.

We investigated the quantitation of oxidative chemical modifications, such as thiol oxidation and carbonylation, in medical-grade human serum albumin (HSA) preparations, in comparison with those of healthy and diseased subjects. Four kinds of HSA products were obtained from three major suppliers in Japan. Eight male collegiate students and six healthy male volunteers were recruited as the young (21.6 years) and older (57.2 years) groups, respectively. Four male stable patients (64.3 years) treated with regular hemodialysis (HD) also enrolled in this study. Quantitative analyses for thiol oxidation and carbonylation were performed using HPLC and spectroscopic methods, respectively. Structural characterization was further investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) spectropolarimetry. Significantly larger amounts of thiol-oxidized and carbonylated HSA products were observed than HSA obtained from healthy subjects. In the structural characterization, the midpoint temperature of the denaturation curve (Tm) analyzed by DSC was relatively high, and may have been caused by the added albumin-specific stabilizers, and CD-resolved secondary structure showed that HSA products had a helical conformation. Commercial HSA products for clinical use have a more thermally stable state and remain in a helix-rich structure, even though their specific amino acids (mainly Cys and Lys residues) are oxidatively modified.

Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor ß chain polymorphic proteins.

The high-affinity IgE Fc receptor (FcεRI) ß chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI ß have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI ß polymorphic (ß-L172I, ß-L174V, and ß-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI ß. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI ß (ß-WT) and ß-E228G. These results suggested that ß-E228G affected the thermal stability of FcεRI ß. The role of ß-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of ß-E228G may affect the function of FcεRI ß.

Elevated oxidative stress monitored via the albumin-thiol redox state is correlated with matrix metalloproteinase-3 elevation in patients with rheumatoid arthritis.

BACKGROUND: In rheumatoid arthritis (RA), matrix metalloproteinase-3 (MMP-3) and oxidative stress contribute to joint destruction. However, little is known about the relationship between MMP-3 and oxidative stress in RA. METHODS: We measured the albumin-thiol redox state as a marker of oxidative stress, MMP-3, and the DAS-28 score calculated using CRP values among forty-seven patients (9 males and 38 females) with RA. According to the serum MMP-3 levels, they were divided into two groups (group A: within normal ranges of 36.9-121.0 ng/mL for men and 17.3-59.7 ng/mL for women; group B: above normal ranges). RESULTS: The albumin-thiol redox state in group B was significantly oxidized compared with that in group A (p < 0.01). The percentage of oxidized albumin-thiol showed a positive correlation with serum MMP-3 (r = 0.52). DAS-28 and CRP were also correlated with the percentage of oxidized albumin-thiol (r = 0.46, r = 0.44). CONCLUSIONS: The albumin-thiol redox state was significantly oxidized in correlation with serum MMP-3 elevation in RA.

Close relationship between redox state of human serum albumin and serum cysteine levels in non-diabetic CKD patients with various degrees of renal function.

AIMS: Human mercaptalbumin (HMA) acts as a covalent-carrier protein for sulfur-containing amino acids, cysteine, and glutathione. In addition, its sulfhydryl residue reacts with peroxyl radicals. In this study, we evaluated the redox state of human serum albumin and its relationship with serum amino thiol levels in non-diabetic chronic kidney disease (CKD) patients with various degrees of renal function. MATERIALS AND METHODS: High performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring were used to analyze serum samples collected from 37 non-dialysis patients with primary glomerulonephritis without diabetes mellitus (DM) or other systemic diseases (non-diabetic CKD patients). RESULTS: Total cysteine and homocysteine plasma levels increased with decreasing renal function and showed a significant negative correlation with glomerular filtration rate. The protein-bound ratio of serum cysteine also changed with the degree of renal dysfunction. The HPLC fraction of human mercaptalbumin (HMA) (%) was significantly lower in nondiabetic CKD patients than in healthy subjects. The redox state of human serum albumin (i.e., HMA %) correlated significantly with the total serum cysteine level. CONCLUSION: The HPLC fraction of HMA (%) closely correlated with the serum cysteine level in non-diabetic CKD patients. An increase in oxidized cysteine with impaired renal function and a reduced plasma redox capacity associated with a decrease in the reduced form of serum albumin (HMA %) may be important risk factors for promoting long-term complications in patients with renal dysfunction.

A lower level of reduced albumin induces serious cardiovascular incidence among peritoneal dialysis patients.

BACKGROUND: Human serum albumin is composed of human mercaptoalbumin (HMA) with cysteine residues having reducing powers and of oxidized human non-mercaptoalbumin. Previously, we reported that a lower HMA level is closely related to serious cardiovascular disease (CVD) incidence and mortality among hemodialysis patients. However, the relationship between HMA level and CVD incidence among peritoneal dialysis (PD) patients is unclear. METHODS: We measured the redox state of human serum albumin using high-performance liquid chromatography in 30 continuous ambulatory PD patients. The association between HMA and incidental CVD events was evaluated. RESULTS: Eight patients experienced symptomatic CVD events (5 patients died) at the 5-year follow-up. The concentration and fraction of HMA (cHMA and f(HMA), respectively) showed significantly lower values in patients with CVD than those without CVD (cHMA 1.58 ± 0.39 and 2.16 ± 0.43 g/dL, f(HMA) 48.9 ± 5.4 and 56.4 ± 8.6%, respectively). Multiple forward stepwise regression analysis using cHMA and f(HMA) as the criterion variables was performed, and C-reactive protein and hemoglobin were adopted as significant explanatory variables in the former equation, whereas urea nitrogen was adopted in the latter equation. Multiple logistic regression analysis revealed that cHMA is a statistically, and f(HMA) is a marginally significant explanatory variable of CVD incidence (p = 0.0369, R = -0.260 and p = 0.0580, R = -0.214, respectively). CONCLUSIONS: Lower HMA level, which might be caused by chronic inflammation, anemia and accumulation of dialyzable uremic toxin(s), is closely related to serious CVD incidence among PD patients.

Plk1 is negatively regulated by RNF8.

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with Plk1.

FcεRI-induced mast cell cytokine production critically involves an aspartic acid residue (D234) in the C-terminal intracellular domain of the FcεRIß chain.

The high affinity IgE Fc receptor (FcεRI) ß chain is well implicated as a signal amplifier through the immunoreceptor tyrosine-based activation motif (ITAM) in its C-terminal intracellular region. Our previous study, however, demonstrated that mutation in all of the three tyrosine residues within the FcεRIß ITAM did not impair FcεRI-induced cytokine production, suggesting a possible functional region other than the ITAM. To investigate the ITAM-independent mechanism by which FcεRIß regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIß mutants were generated. We observed that truncation of the FcεRIß C-terminus downstream of the ITAM resulted in a considerable decrease in FcεRI-induced IL-6 production but not degranulation. Furthermore, mutagenesis of a single C-terminal aspartic acid (D234) to alanine (ß-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and ß-D234A suggests that the secondary structure of the FcεRIß C-terminus was not affected by the D234A mutation. Consistently, we did not observe any effect of this mutation on FcεRI-induced tyrosine phosphorylation of FcεRIß. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIß ITAM.

Comparative intermolecular cross-relaxation studies of human hemoglobin in red blood cells and bovine serum albumin in solution.

Intermolecular cross-relaxation rate (CR) spectra [1/T(IS) (HDO) or 1/T(IS) (H(2) O) vs f(2) (ppm) profiles] for bovine serum albumin [BSA; molecular weight (MW), 66 kDa] solution, partially hydrolyzed BSA gel (BSA*gel) and packed human red blood cells (RBCs) with normal or unstable hemoglobin (Hb; MW, 65 kDa) were studied using f(2) irradiation ranging from - 100 to 100 ppm at γH(2) /2π of 250 Hz. The CR spectra for BSA*gel (pD 4.01, 0.10 M NaCl, 4.83 and 14.39%) exhibited different features in the off-resonance region (below - 2.00 and above 12.0 ppm) relative to that for BSA solution (pD 7.14, 0.10 M NaCl, 14.39%), indicating the association of BSA* molecules in the gel state. The CR spectrum for packed RBCs was compared with those for BSA*gel and BSA solution (14.39%) by correcting for differences in protein concentration. The corrected CR spectrum for packed normal RBCs in the off-resonance region was similar to that for BSA solution, indicating that the physical characteristics of Hb in normal RBCs may be in a solution-like state. Our results on normal RBCs were approximately consistent with the previously reported thermodynamic and hydrodynamic findings that Hb in RBCs and/or in concentrated solution seems to be in a suspension of hard scaled particles.

Comparative 1H NMR studies of saturation transfer in copolymer gels and mouse lenses.

Saturation transfer in cross-linked copolymer gels and excised intact and perforating trauma-induced cataract mouse lenses (4- or 8-week-old) were studied using intermolecular cross-relaxation rates (1/T(IS)(H(2)O); 1/T(IS)), monitored with f(2)-irradiation at -8.79, -4.00, and 7.13 ppm (gammaH(2)/2pi approximately 69 Hz). [1] The 1/T(IS)(7.13 ppm) vs dry weight [W (%)] profiles for hydrophilic copolymer gels were far steeper than those for hydrophobic copolymer gels, indicating the participation of an amount of bound water and a number of copolymer hydroxyl groups in the saturation transfer process. In contrast, the 1/T(IS)(-8.79 ppm) vs W (%) profiles exhibited little difference between the hydrophilic and hydrophobic copolymer gels, indicating the major participation of molecular rigidity, i.e. W (%) in the saturation transfer process. [2] The 1/T(IS)(7.13 ppm) values for cataractous mouse lenses were larger than those for intact lenses, indicating the formation of large, immobile lens protein associates or aggregates containing a sufficient amount of bound water for the saturation transfer. [3] The 1/T(IS)(7.13 ppm) vs W (%) profiles for the hydrophilic copolymer gels exhibited similar characteristics to the intact and cataractous mouse lenses with regard to the saturation transfer process.

Albumin thiol oxidation and serum protein carbonyl formation are progressively enhanced with advancing stages of chronic kidney disease.

BACKGROUND: Oxidative stress is enhanced in advanced chronic kidney disease (CKD) patients and recognized as a main contributor to cardiovascular disease. Carbonyl stress is also known to be enhanced in advanced CKD; however the precise relationship between oxidative stress and carbonyl stress is not clear. The aim of this study was to investigate potential relationships between oxidative stress, carbonyl stress, and renal function among predialysis patients with CKD. METHODS: A total of 32 predialysis CKD patients (22 male, 10 female) were divided into four groups according to their values for creatinine clearance (Ccr) (group A, > or =60 ml/min; group B, 45-59 ml/min; group C, 30-44 ml/min; group D, < or =29 ml/min). As main markers of oxidative and carbonyl stresses, the redox state of Cys-34 (free thiol group) of human serum albumin [HSA(Cys-34)-redox] and the carbonyl content of serum proteins were employed, respectively. RESULTS: The values for the fraction of both reversibly oxidized HSA [f(HNA-1)] and irreversibly oxidized HSA [f(HNA-2)] significantly increased with a decrease in renal function (group A, 21.0 +/- 3.4 and 1.8 +/- 0.3%; group D, 31.1 +/- 4.1 and 2.7 +/- 0.9%, respectively). The value for carbonyl content also significantly increased with a decrease in renal function (group A, 0.7 +/- 0.1 nmol/mg protein; group D, 1.1 +/- 0.2 nmol/mg protein). There was a significant positive correlation between carbonyl content and the f(HNA-2) value, while such a correlation was not observed between carbonyl content and the f(HNA-1) value, suggesting that there is a close relationship between serum protein carbonylation and irreversible albumin thiol oxidation. CONCLUSIONS: There is a close relationship between oxidative stress and carbonyl stress and these are enhanced in correlation with the level of renal dysfunction among predialysis CKD patients.

Comparative 1H NMR studies on the structural looseness of the aged (A) and non-aged (N) bovine mercaptalbumin in the alkaline region.

Bovine serum albumin (BSA) and mercaptalbumin (BMA) exhibit the N-B transition in the alkaline region (pH 7.0-->9.0). BMA has 17 disulfide bonds and one SH group at Cys-34 which catalyzes the intramolecular SH, S-S exchange reaction at low ionic strength in the alkaline region (the N-A isomerization, where N and A indicate the non-aged and aged BMA, respectively). The N-A isomerization is a reversible first-order reaction of the type N<-->A. In the alkaline region, such as pH 8.6, (A)/(N) was approximately 1 in the absence of added salt and approximately 0 in 0.10 M NaCl, indicating the shift of the equilibrium from the A- to the N-forms. Using iodoacetamide-blocked BSA (IA-BSA) and aged BMA (IA-A-BMA), the 1H NMR cross-relaxation times (TIS), which reflect the structural looseness in proteins, were measured in the N-, B- (IA-BSA), N*- and B*-forms (IA-A-BMA), where the N*- and B*-forms indicate IA-A-BMA in the neutral (approximately pD 7) and alkaline regions (approximately pD 9), respectively. At pD 9.1-9.4 in the absence of added salt, the TIS values for the B- and B*-forms exhibited elongation, indicating the liberation of structural looseness resulting in (A)/(N) approximately 1. At pD 9.3 in 0.10 M NaCl, the TIS values for the B*-form exhibited elongation and those for the B-form did not, indicating the presence of structural looseness in only the B*-form resulting in (A)/(N) approximately 0.

[Clinical evaluation of serum albumin reductivity in patients with renal dysfunction: a comparison between conservative renal failure patients and hemodialysis patients].

We examined the relationship between change in the redox state of the plasma albumin molecule and the metabolic disorder of sulfur amino acid observed being accompanied by reduction of renal function. Thirty-seven cases of pre-dialysis renal failure with conservative treatment and thirteen cases of chronic hemodialysis were selected as the subjects of this examination. The fraction of plasma albumin and the concentration of plasma cysteine and homocysteine were respectively measured by the HPLC and GC/MS (gas chromatography/mass spectrometry) methods. In the case of pre-dialysis renal failure with conservative treatment, the reduction rate of plasma albumin significantly decreased in correspondence with reduction of the glomerular filtration rate (GFR). It is well known that the reduction rate of plasma albumin also decreases with the aging process. However, in regard to chronic hemodialysis, a correlation with aging was not found, where the transient reduction rate of plasma albu- min increased after the hemodialysis session. However, in correspondence with the decrease in renal function, the concentration of plasma cysteine and homocysteine increased. This shows that there was a negative correlation with GFR in cases of pre-dialysis renal failure with conservative treatment. In cases of chronic hemodialysis, the concentration of free cysteine and free homocysteine rapidly decreased after a hemodialysis session. Therefore, a negative correlation was recognized between the reduction rate of plasma albumin and the concentration of plasma cysteine and homocysteine. The result of this examination shows the following mechanisms: plasma albumin plays an important role in the reaction of oxidation/reduction in blood plasma, and sulfur amino acid in blood plasma, especially the abnormality of cysteine concentration, plays an important role in changing the redox state of the blood plasma observed in the decrease in renal function.

Oral branched-chain amino acid supplementation improves the oxidized/reduced albumin ratio in patients with liver cirrhosis.

AIM: Branched-chain amino acid (BCAA) supplementation improves hypoalbuminemia in decompensated cirrhotics. Recently, it was clarified that the ratio of oxidized albumin within total albumin rises with progression of liver cirrhosis. We conducted a feasibility study to investigate whether BCAA supplementation might improve this ratio. METHODS: Seven cirrhotic patients (age: 70 +/-> 6 years; M/F = 4/3; etiology: hepatitis C in six and non-B/non-C hepatitis virus in one; Child-Pugh classification: A in six and B in one) were enrolled consecutively in this study in October 2004 to March 2005. Patients were given 4 g BCAA after each meal for 8 weeks. Serum total, oxidized and reduced albumin, plasma amino acids, glutathione, zinc, selenium, and lipid peroxide concentrations were measured every 2 weeks. RESULTS: Low total albumin, high oxidized albumin, and low reduced albumin levels were observed at entry. After 8 weeksBCAA supplementation, the ratio of oxidized albumin within total albumin decreased significantly and that of reduced albumin increased significantly (P < 0.05, respectively). Total albumin tended to rise and lipid peroxide concentrations tended to fall, but not significantly. CONCLUSION: BCAA supplementation improved the oxidized/reduced state of serum albumin. This intervention is effective to maintain the quality of serum albumin in cirrhotic patients.

Dialyzable uremic solutes contribute to enhanced oxidation of serum albumin in regular hemodialysis patients.

BACKGROUND: Oxidative stress (OS) is reportedly enhanced in patients receiving regular hemodialysis (HD). However, the in vivo redox state of HD patients, particularly after HD sessions, remains unclear. This study aimed to clarify the influence of HD on OS using the albumin redox state as a marker. METHOD: Blood samples of 8 regular HD patients were obtained during the course of study. The redox state of albumin was determined using high-performance liquid chromatography. RESULTS: The mean fraction of reversibly oxidized albumin [f(HNA-1)] declined significantly over the course of the session and reached a minimum 4 h after the session had ended (pre-HD, 36.16 +/- 7.50%; 4 h after HD, 25.71 +/- 6.41%), then gradually rose to predialytic levels. The proportion of irreversibly oxidized albumin did not change significantly over time. Positive correlations were demonstrated between f(HNA-1) and uremic small solutes in each case. CONCLUSION: Accumulation of dialyzable uremic solutes may contribute to OS in HD patients.

Equivalent cross-relaxation rate imaging and diffusion weighted imaging for early prediction of response to bevacizumab-containing treatment in colorectal liver metastases-preliminary study.

PURPOSE: To evaluate and compare the usefulness of equivalent cross-relaxation rate (ECR) imaging (ECRI) and diffusion-weighted imaging (DWI) in the early prediction of the response of bevacizumab-containing treatments of colorectal liver metastases. METHODS AND MATERIAL: Seven patients received bevacizumab-containing treatments for colorectal liver metastases. Serial magnetic resonance imaging was performed to evaluate responses before and 2 weeks after starting chemotherapy. In the ECRI, we adopted the off-resonance technique for preferential saturation of immobile protons to evaluate the ECR values. A single saturation transfer pulse frequency was used at a frequency of 3.5 ppm downfield from the water resonance. In the DWI, the apparent diffusion coefficient (ADC) value commonly used with two b-values was acquired by using diffusion weightings of 0 and 800 s/mm2. The region of interest of the metastatic lesions in the liver was separately measured by ECRI and DWI. Tumor response was assessed by response evaluation criteria in solid tumors criteria 8 weeks after starting chemotherapy. RESULTS: In this study, we had four responders and three nonresponders. There was a significant difference in the pretreatment ECR values between the responders and nonresponders (P=.01); there was no significant difference in the ADC values between the two groups. Analysis of the percentage difference between the pretreatment and post-treatment values, termed as percentage change, showed that there were no significant differences in the percentage change of the ADC values between both groups; however, the percentage change in the ECR value was significantly greater for the responders than for the nonresponders (-41.6%±17.1% vs. -12.9%±6.9%, respectively; P=.04). CONCLUSION: The pretreatment ECR value and percentage change of the ECR value 2 weeks after starting chemotherapy were useful parameters in the early prediction of response to bevacizumab-containing treatment in colorectal liver metastases.

An event-related potential investigation of sentence processing in adults who stutter.

The purpose of this study was to investigate characteristics of the semantic processing of sentences' final verbs in stutterers using event-related potential (ERP). ERPs elicited from semantically violating and non-violating verbs in Japanese sentences were compared between 13 adults who stutter (AWS) and 13 adults who do not stutter (AWNS). The stimulus sentences elicited the N400 and the late positive component (LPC) in both groups. The amplitude of the N400, however, was attenuated in AWS. Regarding the LPC, the LPC in the 450-700ms time window (the early LPC) was evident in both groups, but the LPC in the 700-850 time window (the late LPC) was only apparent in AWS. Because AWS judged sentence congruency as accurately as AWNS did, it is assumed that AWS depended more on the LPC for semantic processing, resulting in the enhancement of the late LPC. We speculate that semantic processing of sentences for AWS is more time consuming than that for AWNS.

Useful markers for detecting decreased serum antioxidant activity in hemodialysis patients.

This study evaluates a novel application of a method for measuring serum antioxidant activity, based on the detection of erythrocyte membrane lipid peroxidation in cases of uremia. A human erythrocyte ghost membrane in Tris-HCl was mixed with adenosine 5'-diphosphate and iron chloride (FeCl3; ADP/Fe3+) solution (at a molar ratio of 17:1), and the mixture was incubated under aerobic conditions at 37 degrees C for 2 hours. The concentration of erythrocyte membrane thiobarbituric acid-reactive substances increased proportionally with respect to ADP/Fe3+ concentration, and this increase was inhibited by serum albumin in a dose-dependent manner. In patients undergoing chronic hemodialysis therapy, predialytic sera contained in this reaction mixture were weaker than postdialytic sera in terms of inhibitory effect against erythrocyte membrane lipid peroxidation, whereas serum albumin contents remained at levels equivalent to those of the normal control. A gradual increase in human mercaptalbumin nonmercaptalbumin ratio during hemodialysis treatment might be one of the major factors that leads to the recovery of decreased serum antioxidant activity. We clearly showed that the serum scavenging activity against erythrocyte membrane lipid peroxidation in hemodialysis patients decreases markedly, and this pathological condition is improved by hemodialysis.

The importance of sample preservation temperature for analysis of the redox state of human serum albumin.

BACKGROUND: Human serum albumin (HSA) is a mixture of human mercaptalbumin (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form). METHODS: We have developed a convenient high-performance liquid chromatographic (HPLC) system for the separation of HSA into HMA and HNA, and studied the mercapt<==>nonmercapt conversion (i.e., dynamic change in the redox state) of HSA. Examination of long-term sample preservation temperature on the redox state of HSA is of fundamental importance for analysis of defense systems against oxidants in humans. RESULTS: The HMA fraction of HSA (f(HMA)) was markedly decreased, i.e., the redox state of HSA samples was more oxidized, when they were kept even at -20 degrees C for 170 days. Moreover, the redox states of five commercial HSA products were analyzed and the results were compared with those for normal control subjects. CONCLUSIONS: Surprisingly, marked decreases in f(HMA) value for all commercial HSA products were observed.

Simple and sensitive high-performance liquid chromatographic method for the investigation of dynamic changes in the redox state of rat serum albumin.

Serum albumin is a mixture of mercaptalbumin (reduced form) and non-mercaptalbumin (oxidized form), i.e. a protein redox couple in serum. To investigate dynamic changes in the redox state of rat serum albumin (RSA), we developed a simple and sensitive high-performance liquid chromatographic (HPLC) system using an ion-exchange column with a linear gradient of ethanol concentration. Furthermore, we applied this HPLC system to examine dynamic changes in the redox state of RSA caused by severe oxidative stress such as exhaustive physical exercise. Using this system, we successfully separated RSA to rat mercaptalbumin (MA(r)) and rat non-mercaptalbumin (NA(r)), and also found the best conditions for the clear separation of RSA. In the experiments with exhaustive exercise, mean values for the MA(r) fraction in control and exercise groups were 76.2+/-1.8 and 69.0+/-3.5%, respectively. The MA(r) in the exercise group was significantly oxidized compared with that of the control group (P<0.01). These results suggested that RSA might act as one of the major scavengers in extracellular fluids under severe oxidative stress.